SUMMARY: DNA damage has long been implicated as a driver of aging. DNA damage is very frequent, with estimates of about 100,000 lesions per cell per day in mammals. Such lesions can impact transcription directly, elicit cellular responses, such as apoptosis and cellular senescence, or result in mutations due to errors during repair or replication of a damaged DNA template. Project 3 has been focused on somatic DNA mutations, which can vary from base substitutions to large chromosomal aberrations. This is commonly termed genomic instability, now considered a hallmark of the aging process. Because DNA mutations cannot be repaired (except through cell or organismal death) they accumulate in cells and tissues during aging, which has been empirically confirmed in multiple species, including humans and mouse. Accurate detection and quantitative analysis of DNA mutations in cells and tissues is a challenge due to the very low abundance of de novo mutations in normal somatic cells. We developed methods that allow for the accurate quantitative detection of de novo somatic mutations in normal cells and tissues. In the previous, still ongoing project period, we used one of these methods to compare mutation frequency and spectra in cells from different rodent species after DNA damage (see Progress Report). Genome maintenance capacity has long been implicated in the evolution of species-specific maximum life span. Hence, Project 3 is testing the hypothesis that cells from short-lived species, such as mice, would show more mutations after DNA damage than the same cell type from long-lived species. In this renewal project period, we will specifically test the hypothesis that genome structural variation (Aim 1) and DNA methylation changes (Aim 2) induced by gamma radiation correlate with species-specific life span in rodents. In Aim 3 we will then test if interventions developed by our collaborators in Project 1 and Project 2, based on the longevity mechanisms discovered in long-lived rodents, promote genome and/or epigenome integrity when applied to mice.